The steroid and thyroid hormone superfamily of nuclear receptors is found in mammals and insects and is composed of over 100 known proteins. These receptors fall into at least two functionally distinct categories known as Class I and Class II (Beato, Cell 56:335-344 (1989); Parker, Sem. Cancer Biol. Ser. 1:81-87(1990)). The best studied examples of Class II receptor proteins are Retinoic Acid Receptor (RAR), Vitamin D Receptor (VDR), and Thyroid Hormone Receptor (T3R) and Retinoic X Receptor (RXR). The receptors bind to the 5′ regulatory region of the target gene and, upon binding of a chemical ligand to the receptor, the receptor affects gene expression by interacting with other transcription initiating factors.
In addition to the Class II receptor proteins found in mammals as described above, receptors of similar structure and activity have been identified in insects such as Drosophila melanogaster (Koelle et al., Cell 67:59 (1991); Christianson and Kafatos, Biochem. Biophys. Res. Comm. 193:1318 (1993); Henrich et al., Nucleic Acids Res. 18:4143 (1990)). The ecdysone receptor (EcR) binds the steroid hormone 20-hydroxyecdysone (referred to herein as “ecdysone”) and, when heterodimerized with the product of the ultraspiracle (USP) gene, transactivates gene expression. USP is most homologous to RXRα, and RXR is capable of forming heterodimers with EcR (Thomas et al., Nature 362:471-475 (1993)).
Class II nuclear receptor polypeptides such as EcR are characterized by the presence of five domains: A/B, C, D, E and F (Evans, R. Science 240:889-895 (1988)), wherein “A/B” refers to the transactivation domain, “C” refers to the DNA binding domain, “D” refers to the hinge/linker domain, “E” refers to the ligand binding domain, and “F” refers to the variable C-terminal domain that is present in some receptor polypeptides.
The “A/B” (transactivation) domain comprises one or more amino acid sequences acting as subdomains that, when combined with the DNA binding domain in a receptor polypeptide, affect the operation of transcription factors during preinitiation and assembly at the TATA box. (See generally, Ptashne, Nature 335:683-689 (1988)). The effect of the transactivation domain is to allow repeated transcription initiation events leading to greater levels of gene expression from a target gene. Different transactivation domains are known to have different degrees of effectiveness in their ability to increase transcription inititiation.
The “C” (DNA binding) domain is a sequence of amino acids having certain functional features that are responsible for binding of the receptor polypeptide to a specific sequence of nucleotides, the response elements, present in the 5′ regulatory region of the target gene.
The “D” (hinge/linker) domain is located between the DNA binding domain and the ligand binding domain.
The “E” (ligand binding) domain of the receptor polypeptide provides the means by which the 5′ regulatory region of a target gene is activated in response to the presence of a chemical ligand. The ecdysone receptor (EcR) from Drosophila melanogaster is one example of a receptor polypeptide where complementary chemical ligands have been identified that bind to the ligand binding domain. The steroid hormone ecdysone triggers coordinate changes in tissue development that results in metamorphosis, and ecdysone has been shown to bind to EcR (Koelle et al. Cell 67:59-77 (1991)). The plant-produced analog of ecdysone, muristerone, also binds to the ligand binding domain of EcR. Other chemicals, such as the non-steroidal ecdysone agonists RH 5849 (Wing, Science 241:467-469 (1988)) and tebufenozide, the latter known as the insecticide MIMIC®, also will act as a chemical ligand for the ligand binding domain of EcR.
In some cases it is desirable to control the time or extent of expression of a phenotypic trait in plants, plant cells or plant tissue. An ideal situation is the regulation of expression of such a trait at will, triggered by a chemical that can be easily applied to field crops, ornamental shrubs, etc. One such system of regulating gene expression that can be used to achieve this ideal situation is the steroid and thyroid hormone superfamily of nuclear receptors, such as EcR/Ultraspiracle heterodimerized receptors.
U.S. Pat. No. 5,880,333, incorporated herein by reference, is drawn to a method of controlling gene expression in plants comprising transforming a plant with at least two receptor expression cassettes and at least one target expression cassette. The first receptor expression cassette comprises a nucleotide sequence for a 5′ regulatory region operatively linked to a nucleotide sequence that encodes a first receptor polypeptide operatively linked to a 3′ termination region. The second receptor expression cassette comprises a nucleotide sequence for a 5′ regulatory region operatively linked to a nucleotide sequence that encodes a second receptor polypeptide operatively linked to a 3′ termination region. The first and second receptor polypeptides comprise a first and second ligand binding domain, respectively, which are mutually distinct. The target expression cassette comprises a nucleotide sequence for a 5′ regulatory region operatively linked to a nucleotide sequence that encodes a target polypeptide operatively linked to a 3′ termination region, wherein the 5′ regulatory region of said target expression cassette is activated by said first and second receptor polypeptides in the presence of one or more chemical ligands, whereby expression of said target polypeptide is accomplished. The method is useful for controlling various traits of agronomic importance, such as plant fertility.
However, despite advances such as those described in U.S. Pat. No. 5,880,333, there exists a continuing need to develop new and effective systems for inducible gene expression in plants, including the need to develop novel chimeric insect hormone receptors with increased responsiveness to chemical ligands. Especially desirable would be the development of chimeric class II insect hormone receptors that function in the absence of their normal heterodimerization partners.